1. Field of the Invention
The present invention is direct to the field of vaccinology, and epidemiology. Specifically, the present invention provides method for quantitating recent secreted antigen specific antibodies from lymphocyte supernatant for evaluation of vaccine or antigen induced specific antibody secretion from ex vivo circulating lymphocytes without ex vivo antigen stimulation.
2. Related Art
This technology is a novel method for measuring in vitro antibody secretion from tissue culture of B lymphocytes in the peripheral blood mononucleus cells (PBMC). Instead of enumerating antibody-secreting cells in the ELISPOT assay, this technology quantifies recent antigen specific secreted antibodies from a fixed concentration of PBMC cells rather than accumulative soluble antibodies in the serum after immunization with a vaccine or exposure to antigen(s). The soluble antibodies in the serum is removed in this method.
3. General Purpose
A clinical trial using this technology demonstrated that the post-immunization human PBMC cells secreted antibodies to cholera toxin in the cell supernatants without any in vitro antigen stimulation after an oral vaccination with a killed cholera vaccine which is an antigenic substance, an antigen and a vaccine for a live immune system. Using this invention, the antibody titers are not confounded by the pre-existing accumulative antibodies in the serum from the same volunteers. The new findings allow for quantitatively measuring the antigen specific antibody production of the PBMC culture in post-vaccination or disease infection of recent antigen exposed human or animal blood samples. This method is specifically useful for determination of recent immune response during vaccine trials in endemic areas where the population already has pre-existing serum titers. It could also be used as a diagnostic method for identify in recent infections. Since this invention controls the soluble antibodies, it is more accurate and precise that serum antibody measurement. For immunogenicity evaluation, the invention measures secreting antibodies form recent vaccine or antigen activated B cells only. It reduces the clinical trial testing sample size than trials using serum antibody assays, which have large amount of pre-existing antibodies.
We describe here a novel method for measuring in vitro antibody secretion from the tissue culture of human B lymphocytes in peripheral blood mononuclear cells (PBMC) after oral vaccination with a killed cholera vaccine. Enzyme-linked immunosorbent assay (ELISA) titers of the antibody secreted in the cell supernatant were determined. The validation results demonstrated that human PBMC remained viable and continued to secrete antibodies (total immunoglobulin A and G (IgA and IgG) for up to 4 days of incubation at 37° C. with 5% CO2 in cell cultures. The secreted antibody concentration correlated positively with the PBMC concentration and incubation time in the tissue culture and correlated negatively with the storage time of the whole blood at room temperature. In vitro assay of secreting antibody in the lymphocyte supernatant (i.e., the ALS assay) is capable of the detecting specific antibody response after oral vaccination with a killed whole-cell-plus-B-subunit cholera vaccine (WC-B) in healthy adults in a phase I clinical trial. Postimmunization PBMC secreted antibodies to cholera toxin in the cell supernatants. Antibody production did not require any in vitro antigen stimulation. In the ALS assay, antigen-specific antibody titers of prevaccination samples were barely detectable, whereas serum antitoxin ELISA titers in background of pre-vaccine samples were significantly higher than the ALS titers.
We conclude that, without any in vitro antigen stimulation after vaccination, PBMC secrete antibodies into the supernatants in the ALS assay. This assay can quantitatively measure the antigen-specific antibody production from the PBMC culture in post-vaccination blood samples.
4. Background
Postvaccination immunity is generally assessed via the use of antibodies in serum, but it is impossible to distinguish between recently produced antibodies and preexisting antibodies. Antibody levels in serum do not represent the latest immune responses accurately, because serum antibodies include the accumulated soluble antibodies that were induced by previous exposure to antigens.
Recent antigen exposure of mucosal T and B cells induces proliferation and differentiation of these cells (14, 25). The activated T and B cells circulate through the thoracic duct into the blood and eventually return to common mucosal sites, such as the lamina propria of the intestine, as matured plasma cells (2, 17, 20, 22, 23, 26).
To develop a sensitive surrogate for assaying local immunity, the lymphocytes traveling from local mucosal areas to the systemic blood circulation are used by methods for in vitro laboratory evaluations such as ELISPOT (6-10, 12, 15, 21; P. W. Lowry, L. M. McFarland, and H. K. Threefoot, Letter, J. Infect. Dis. 154:730, 1986). In its final step, ELISPOT measures the results of specific antibody-secreting cells (ASC) on a spot-forming gel (11-13, 15, 18; Lowry et al., letter). ELISPOT measures the number of antibody producing cells per 106 PBMC following oral vaccination (11, 16). The quantification of antibodies secreted by a fixed concentration of PBMC is as important as the enumeration of ASC.
This invention is a novel method for measuring in vitro secreting antibody from human lymphocyte's supernatant, i.e., the ALS assay, which directly measures antibody secretions from PBMC of peripheral blood on a micro titer plate.
The ALS assay has been validated by the measurement of total immunoglobulin A (IgA) and IgG production under a series of tissue culture conditions (PBMC inoculation concentration, incubation time, and blood storage time). Then, 107 PBMC was used to determine the antigen-specific antibodies to cholera toxin after the oral vaccination of a licensed Vibrio cholerae vaccine in a phase I clinical trial.
Two formulations of a killed whole-cell-plus-B-subunit cholera vaccine (WC-B) were used to immunize 12 healthy adults. A standard liquid formulation of the vaccine was stored continuously at 4° C., and a spray-dried formulation of the vaccine was placed at room temperature for 30 days. Volunteers were randomized to receive two doses of either vaccine in a double-blind manner. The vaccine induced an elevation in cholera toxin-specific antibodies in sera and induced secretive toxin-specific antibodies in the ALS assay. The ALS assay is potentially an accurate surrogate for measuring recent antibody response and for the diagnosis of recent infections in humans.